Current Genomics - Volume 4, Issue 2, 2003

Human osteoarthritis (OA) is a complex multi-factorial disease that primarily targets the cartilage. The cartilage is a specialized tissue where the chondrocytes maintain matrix homeostasis. The paradigm for the post genomic era of molecular medicine is to identify the function of defective genes associated with specific diseases. Although the process of identification and validation of target genes is feasible with the available tools and technologies, establishing the link between defected gene products and the disease is still a challenging task. New tools in genomic analysis not only allow us to profile gene expression in normal and diseased tissues, but also facilitate the development of unbiased hypothesis at the molecular level. Several defective heritable genes encoding the matrix components (such as Col2a1 and Col9a1 that code for type II and IX collagen respectively) have been linked to the development of OA in human and animal models. Several families of genes expressed differentially in normal and osteoarthritis-affected cartilage have been identified. These include matrix metalloproteinases (MMPs), matrix proteins, integrins, transcription factors, cytokines, cytokine receptors, growth factors and homeostatic genes. A non-conventional functional genomic approach is required to study the complexity of chondrocyte and matrix interactions that affect the above mentioned targets.

Joubert syndrome (JS) is a rare autosomal recessive syndrome characterised by agenesis or dysgenesis of the cerebellar vermis with accompanying brainstem malformations (OMIM: 213300). There is a distinctive (but not unique) MRI appearance in the posterior fossa of the brain termed the molar-tooth-sign, and the diagnosis of JS is usually made when this MRI finding is documented in an infant in combination with hypotonia, episodic hyperpnea or apnea, abnormal eye movements, with a variety of facial and other dysmorphisms. Later, developmental delay and cerebellar ataxia develop. In some JS patients a retinal dystrophy, coloboma and renal abnormalities manifest. It is apparent that JS is a clinically heterogeneous entity and that both intrafamilial and interfamilial variation exists. Overlap with other vermal hypoplasias and rare cerebellar-retinal-renal disorders exists, but whether such disorders are allelic to forms of JS awaits gene isolation and molecular clarification. Recently the first JS locus was found on chromosome 9q34 by homozygosity mapping in a consanguineous family of Omani origin. A second consanguineous family of similar ethnicity demonstrated haplotype segregation consistent with linkage to this 9q34 locus. Linkage to the 9q34 region was excluded in two additional consanguineous families, establishing genetic heterogeneity in the Joubert syndrome. Haplotype analyses in an additional 26 Joubert pedigrees including three with consanguinity, showed no evidence of homozygosity within this interval. These data suggest that the major JS genetic loci are yet to be discovered. Genes crucial to cerebellar and brainstem development are functional candidates, particularly homeotic genes specifying the vermal domain. We have excluded several strong functional candidate genes such as WNT1, EN1, EN2 and FGF8, by mutation analysis and direct DNA sequencing. To characterize the underlying molecular mechanism of the JS, it may be necessary to further define the syndrome. By careful examination, the JS can be distinguished from the related but genetically distinct syndromes, COACH, Senior-Loken and Arima.

Today, in various aspects of molecular biology, sequence alignment has become an essential tool to study the structure-function relationships of proteins. With the impressive increase of the number of available sequences, alignments provide a substantial piece of information by way of various computational methods. These approaches have generally become a crucial tool to put forward working hypotheses for time-consuming bench work, as protein engineering and site directed mutagenesis. However alignment methods remain hugely perfectible. All methods are dramatically limited in the twilight zone, taking place around 25% of identity between pairs of sequences. More worrying is the very high rate of false positive results generated by most algorithms, depending of empirical parameters, and hard to validate by statistical criteria.After reviewing the main methods, this paper draws user's attention to the fact that algorithm performance evaluations are entirely limited to alignment power (sensibility) evaluation. In reference to a given truth defined from alignment of know structures, the power is defined as the proportion of truth restored in the solution. The power may be overestimated by a lack of independent sets of poorly related sequences and its value depends entirely on the criterion used to define the truth. On the other hand, confidence (selectivity) represents the proportion of the solution that is true. Depending on the method and the parameters used, confidence may be much lower than power, and is usually never evaluated. For non-trivial alignments, when the power is high, confidence is low, which means that correctly aligned positions are embedded in large regions unduly aligned.One possible solution to these problems is to use consensus of several multiple alignment methods, which will increase the confidence of the results. The addition of external information, such as the prediction of the secondary structure and / or the prediction of solvent accessibility is also an other way that should increase the performance of existing multiple alignment methods.

Kallikreins are a subgroup of serine proteases with diverse physiological functions. The human kallikrein gene family has now been fully characterized and includes 15 members tandemly located on chromosome 19q13.4 In this review, we discuss the common structural features of kallikreins at the DNA, mRNA and protein levels and summarize their tissue expression and hormonal regulation. Kallikreins are expressed in a wide range of tissues including the salivary gland, endocrine tissues including testis, prostate, breast, endometrium, and the central nervous system. Most, if not all genes are under steroid hormone regulation. The classical kallikreins (KLK1-3) are thought to represent a distinct evolutionary subgroup of kallikreins. The occurrence of several splice variants is a very common phenomenon among kallikreins, and some of the splice variants appear to be tissue-specific and might be related to certain pathological conditions. We also provide a summary of predicted and experimentally confirmed promoter elements of kallikrein genes and describe repeat elements and polymorphisms within this genomic region.

Targeting of the therapeutic genes to the malignant or non-malignant tissue may be achieved using recombinant viral vectors for the transfer and tissue-specific or tumor-specific regulatory sequences. However, the efficiency of the recombinant viruses to infect tumor cells, was shown to be rather low. Therefore, repeated injections of the virus might be required to obtain a sufficient therapeutic response and in clinical trials non-invasive procedures have to be employed to assess the transfer and functional activity of the recombinant gene.In gene therapy based on the transfer and expression of the suicide genes usually genes coding for the non-mammalian enzymes, the Herpes simplex virus thymidine kinase (HSVtk) or the yeast and bacterial cytosine deaminase (CD), have been applied. After infection of the tumor with the recombinant virus, the non-toxic prodrug is applied systemically, which is subsequently converted to a toxic metabolite by the recombinant gene product. Employing a radiolabeled prodrug and scintigraphic procedures to determine the functional activity of the recombinant enzyme in vivo, a therapeutic window of maximal gene expression and consecutive drug administration may be defined. If the gene therapy approach is based on the transduction of receptor genes, the recombinant gene expression in tumor cells will be monitored using radiolabeled ligands. Transfer of transporter genes as the sodium iodide transporter may also lead to the visualization of transduction via accumulation of iodide or pertechnetate. Furthermore, imaging based on transchelation of oxotechnetate to a polypeptide motif from a biocompatible complex with a higher dissociation constant than that of a dicglycilcysteine complex or tyrosinase gene transfer for metal ion scavenging have been described.In addition, the monitoring of the gene therapy effects is performed by the evaluation of the morphological changes of the tumor using magnetic resonance imaging or, more effectively, by the measurement of the tumor-associated metabolic variations with positron emission tomography (PET) employing tracers of tumor metabolism and proliferation. The uptake of 18Fluordeoxyglucose (FDG) has been demonstrated to be a useful parameter for the assessment of the glucose metabolism and the tumor growth may be elevated by the uptake of (11C)thymidine or other proliferative markers.

Reverse transcription polymerase chain reaction (RT-PCR) techniques are increasingly used for the quantification of gene transcripts. Compared to other methods permitting relative or absolute quantitation of mRNA, the RT-PCR approach offers the advantages of superior sensitivity, high specifity, and great versatility. In this review, we discuss different approaches to PCR-based quantification of gene expression including comparative, competitive, and real-time PCR. Different formats of fluorescence-based real-time PCR and their applications in clinical diagnosis and research are presented.